Mutations in the dystrophin gene result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). There is no cure for these devastating diseases. An effective therapy relies on treating all affected muscles in the body. Adeno-associated virus (AAV) can efficiently deliver a therapeutic gene to all body muscles. Therefore, replacing the defective dystrophin gene with AAV gene therapy holds great therapeutic promise. However, AAV vector has a 5 kb packaging capacity. This is insufficient to deliver the 11.06 kb full-length dystrophin coding sequence. For this reason, current AAV gene therapy is forced to use the massively truncated mini- or micro-dystrophin genes. In this application, we propose to develop a novel tri- AAV system to express the full-length protein. The combined capacity of three AAV vectors provides enough room for the full dystrophin coding sequence. However, random recombination among different AAV vectors will lead to very inefficient reconstitution of the full-length protein. We, among others, have recently developed a series of novel approaches to facilitate unidirectional recombination between different AAV vectors. In this project, we will apply these new technologies to the development of the tri-AAV full-length dystrophin vectors. Our specific aims are: (1) to design and construct full-length dystrophin tri-AAV vectors, and (2) to evaluate the full-length dystrophin tri-AAV vectors in dystrophin-deficient mdx mice by local gene transfer. We believe that this high payback translational project will likely lead to a breakthrough in AAV-mediated DMD gene therapy in the future. Duchenne muscular dystrophy (DMD) is a life threatening disease affecting a fairly large population (more than one in 3,500 newborns). It is caused by dystrophin gene mutations. The best therapy is to replace the mutated gene with a normal gene. AAV vector can express truncated versions of the dystrophin gene but it cannot express the full-length protein. In this translational project, we propose to develop a novel tri-AAV system to express the full-length dystrophin protein. If successful, this project will lead to a big breakthrough in DMD gene therapy.